WO2016199003A1 - Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof - Google Patents
Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof Download PDFInfo
- Publication number
- WO2016199003A1 WO2016199003A1 PCT/IB2016/053265 IB2016053265W WO2016199003A1 WO 2016199003 A1 WO2016199003 A1 WO 2016199003A1 IB 2016053265 W IB2016053265 W IB 2016053265W WO 2016199003 A1 WO2016199003 A1 WO 2016199003A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polysaccharide
- protein
- atleast
- conjugate
- adsorption
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 99
- 238000009472 formulation Methods 0.000 title claims abstract description 55
- 238000001179 sorption measurement Methods 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 48
- 229940031348 multivalent vaccine Drugs 0.000 title description 3
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 166
- 239000005017 polysaccharide Substances 0.000 claims abstract description 166
- 150000004676 glycans Chemical class 0.000 claims abstract description 152
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 58
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 58
- 238000004220 aggregation Methods 0.000 claims abstract description 38
- 230000002776 aggregation Effects 0.000 claims abstract description 38
- 108010060123 Conjugate Vaccines Proteins 0.000 claims abstract description 14
- 229940031670 conjugate vaccine Drugs 0.000 claims abstract description 14
- 239000001384 succinic acid Substances 0.000 claims abstract description 9
- 230000007935 neutral effect Effects 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 230000002378 acidificating effect Effects 0.000 claims abstract description 4
- 239000007853 buffer solution Substances 0.000 claims abstract description 4
- 102000014914 Carrier Proteins Human genes 0.000 claims description 44
- 108010078791 Carrier Proteins Proteins 0.000 claims description 44
- 229960005486 vaccine Drugs 0.000 claims description 26
- 108010071134 CRM197 (non-toxic variant of diphtheria toxin) Proteins 0.000 claims description 24
- 101710087657 Manganese ABC transporter substrate-binding lipoprotein Proteins 0.000 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 230000021615 conjugation Effects 0.000 claims description 20
- 238000006731 degradation reaction Methods 0.000 claims description 18
- 230000015556 catabolic process Effects 0.000 claims description 17
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 15
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 15
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 15
- 229940068977 polysorbate 20 Drugs 0.000 claims description 15
- 239000002671 adjuvant Substances 0.000 claims description 13
- 229940031999 pneumococcal conjugate vaccine Drugs 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 10
- 159000000013 aluminium salts Chemical class 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 230000001404 mediated effect Effects 0.000 claims description 7
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 claims description 6
- 241000193998 Streptococcus pneumoniae Species 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 229940031000 streptococcus pneumoniae Drugs 0.000 claims description 6
- 229960000814 tetanus toxoid Drugs 0.000 claims description 6
- 229940037003 alum Drugs 0.000 claims description 5
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 claims description 5
- 229910000329 aluminium sulfate Inorganic materials 0.000 claims description 5
- 241000193738 Bacillus anthracis Species 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 101710194807 Protective antigen Proteins 0.000 claims description 4
- 229940065181 bacillus anthracis Drugs 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 229960003983 diphtheria toxoid Drugs 0.000 claims description 4
- 230000005847 immunogenicity Effects 0.000 claims description 4
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 150000004713 phosphodiesters Chemical class 0.000 claims description 4
- 108010040473 pneumococcal surface protein A Proteins 0.000 claims description 4
- 239000003755 preservative agent Substances 0.000 claims description 4
- 229940070741 purified protein derivative of tuberculin Drugs 0.000 claims description 4
- -1 19A polysaccharide Chemical class 0.000 claims description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 3
- 239000006227 byproduct Substances 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000003118 sandwich ELISA Methods 0.000 claims description 3
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 3
- QCDWFXQBSFUVSP-UHFFFAOYSA-N 2-phenoxyethanol Chemical compound OCCOC1=CC=CC=C1 QCDWFXQBSFUVSP-UHFFFAOYSA-N 0.000 claims description 2
- 102100037840 Dehydrogenase/reductase SDR family member 2, mitochondrial Human genes 0.000 claims description 2
- 101710082714 Exotoxin A Proteins 0.000 claims description 2
- 241000606768 Haemophilus influenzae Species 0.000 claims description 2
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 2
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 2
- 102000018697 Membrane Proteins Human genes 0.000 claims description 2
- 108010052285 Membrane Proteins Proteins 0.000 claims description 2
- 206010030113 Oedema Diseases 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- 201000005702 Pertussis Diseases 0.000 claims description 2
- 101710183389 Pneumolysin Proteins 0.000 claims description 2
- 102000017033 Porins Human genes 0.000 claims description 2
- 108010013381 Porins Proteins 0.000 claims description 2
- 101710188053 Protein D Proteins 0.000 claims description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 2
- 101710132893 Resolvase Proteins 0.000 claims description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 2
- 102000010912 Transferrin-Binding Proteins Human genes 0.000 claims description 2
- 108010062476 Transferrin-Binding Proteins Proteins 0.000 claims description 2
- 235000019445 benzyl alcohol Nutrition 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000012634 fragment Substances 0.000 claims description 2
- 231100000518 lethal Toxicity 0.000 claims description 2
- 230000001665 lethal effect Effects 0.000 claims description 2
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
- 229960005323 phenoxyethanol Drugs 0.000 claims description 2
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 claims description 2
- 238000004513 sizing Methods 0.000 claims description 2
- 239000004094 surface-active agent Substances 0.000 claims description 2
- 229940033663 thimerosal Drugs 0.000 claims description 2
- XLJMAIOERFSOGZ-UHFFFAOYSA-M cyanate Chemical compound [O-]C#N XLJMAIOERFSOGZ-UHFFFAOYSA-M 0.000 claims 4
- 238000002156 mixing Methods 0.000 claims 1
- 230000002335 preservative effect Effects 0.000 claims 1
- 238000010791 quenching Methods 0.000 claims 1
- 230000000171 quenching effect Effects 0.000 claims 1
- 239000000243 solution Substances 0.000 description 23
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 22
- 230000004913 activation Effects 0.000 description 12
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 10
- 238000011026 diafiltration Methods 0.000 description 10
- 239000000427 antigen Substances 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000008215 water for injection Substances 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 6
- 238000004132 cross linking Methods 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000004255 ion exchange chromatography Methods 0.000 description 5
- 208000035109 Pneumococcal Infections Diseases 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000013020 final formulation Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 208000022120 Jeavons syndrome Diseases 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 229940001007 aluminium phosphate Drugs 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 2
- 239000013256 coordination polymer Substances 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 239000012526 feed medium Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000004845 protein aggregation Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000005549 size reduction Methods 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000036962 time dependent Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 201000001178 Bacterial Pneumonia Diseases 0.000 description 1
- 208000034628 Celiac artery compression syndrome Diseases 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 238000003231 Lowry assay Methods 0.000 description 1
- 238000009013 Lowry's assay Methods 0.000 description 1
- 206010027202 Meningitis bacterial Diseases 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000000240 adjuvant effect Effects 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 201000009904 bacterial meningitis Diseases 0.000 description 1
- 201000005008 bacterial sepsis Diseases 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- BBWBEZAMXFGUGK-UHFFFAOYSA-N bis(dodecylsulfanyl)-methylarsane Chemical compound CCCCCCCCCCCCS[As](C)SCCCCCCCCCCCC BBWBEZAMXFGUGK-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 238000002983 circular dichroism Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 206010013023 diphtheria Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003116 impacting effect Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000001738 isopycnic centrifugation Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000569 multi-angle light scattering Methods 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940031960 pneumococcal polysaccharide vaccine Drugs 0.000 description 1
- 229960001973 pneumococcal vaccines Drugs 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 229960002668 sodium chloride Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960004906 thiomersal Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012646 vaccine adjuvant Substances 0.000 description 1
- 229940124931 vaccine adjuvant Drugs 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
- A61K39/092—Streptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/085—Staphylococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/116—Polyvalent bacterial antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
Definitions
- Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis in children. Recent estimates of child deaths caused by 5 pneumoniae range from 0.7 - 1.0 million every year worldwide. In 2000, about 14 ⁇ 5 million episodes of serious pneumococcal disease (uncertainty range 11 ⁇ 1-18-0 million) were estimated to occur. Pneumococcal disease caused about 0.826 million deaths (0.58 -0.926 M) in children aged 1-59 months, of which 0.091 million (0.063-0.1 M) were in HIV-positive and 0.735 million (0.51-0.82 M) in HIV-negative children.
- the multivalent pneumococcal polysaccharide vaccines that have been licensed for many years have proved valuable in preventing pneumococcal disease in adults, particularly, the elderly and those at high-risk.
- infants and young children respond poorly to unconjugated pneumococcal polysaccharides.
- the pneumococcal conjugate vaccine, Prevnar® containing the 7 most frequently isolated serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) causing invasive pneumococcal disease in young children and infants at the time, was first licensed in the United States in February 2000.
- PrevnarTM 13 is an approved vaccine that contains conjugates of polysaccharides from serotypes 6A, 6B, 19A, 19F in addition to 1, 3, 4, 5, 7F, 9V, 14, 18C and 23F.
- SynflorixTM is another approved vaccine that provides protection against 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F as well as cross protection against 19A & 6 A.
- Vaccine formulations must generally be stable and be of uniform consistency to accommodate the need for a long shelf-life and the use of multiple dose containers.
- Vaccines based on proteins, including polysaccharide -protein conjugates are subject to protein aggregation and precipitation which can result in an effective lower total concentration of the vaccine due to the unavailability of the precipitated protein product.
- Polysaccharide -protein conjugate vaccines in particular, appear to have a stronger tendency to aggregate than the carrier protein alone (See Berti et al, 2004, Biophys J 86:3-9).
- the choice of formulation for a polysaccharide -protein conjugate vaccine can greatly affect protein aggregation. See Ho et al., 2001, Vaccine 19:716-725.
- Immunological properties and stability of conjugate vaccine candidate adsorbed on aluminium adjuvants depends on various parameters like i) antigenicity of each antigen, ii) type of carrier protein used for conjugation, and iii) the type of adjuvant used. Most importantly, the extent of adsorption of antigen on the adjuvant has been previously reported to be one of the key parameters to demonstrate the lot to lot consistency of the formulation process and its possible impact on the efficacy of the vaccine product. Further, percent adsorption of polysaccharide-protein conjugates may drop further on storage of the formulation or under the adverse situations like temperature excursions.
- formulate can comprise of aggregates of polysaccharide-polysaccharide type, protein-protein type or polysaccharide-protein type. Such aggregations are also observed in the finished product leading to rejection of 4% to 10% of filled vials of polysaccharide-protein conjugate(s) vaccine, thereby affecting the stability and efficacy of the conjugate vaccine.
- the invention provides improvements in the stability of vaccines which include aluminium salts, and in particular methods for minimizing aggregation and improvements in percent adsorption of individual conjugates in a multivalent pneumococcal polysaccharide -protein conjugate vaccines.
- the inventors of present invention have observed that combined adsorption of polysaccharide -protein conjugates and use of polysaccharide to protein ratio greater than 1 : 1 results in i) percent adsorption of less than 55% for S.
- the instant invention relates to a method for the preparation of a stable multivalent pneumococcal polysaccharide-protein conjugate vaccine formulation, showing optimal adsorption between 75 to 99% for each conjugate wherein, in said method, aggregation is prevented by employing at least one of: a. Individual or separate adsorption for conjugates that otherwise show relative lower adsorption by combined adsorption; b. Histidine-Succinic acid buffer system along with shift in pH from neutral pH to acidic pH; c. A polysaccharide to protein ratio between 0.6 to about 1.4; and/or d. The use of a six bladed Rushton type turbine impeller in the formulation vessel.
- the instant invention also discloses a method for preparing polysaccharide-protein conjugates with improved immunogenicity and less free polysaccharide content for Streptococcus pneumoniae polysaccharides containing phosphodiester linkage, particularly 19A, 19F, 6A and 6B. Said conjugation process minimizes cyanylation agent by-product mediated degradation of sized polysaccharide and prevents subsequent polysaccharide -polysaccharide aggregation thereby stabilizing labile polysaccharides.
- a key to reduced aggregation can be attributed to the use of a sized polysaccharide in the range of 100 - 200 KDa, and polysaccharide to CDAP (Cyanylation agent) ratio in the range of (1):(0.8 - 1).
- the immunogenic composition prepared as per the instant invention provides reduced aggregation between Polysaccharide - Polysaccharide, and Polysaccharide - Protein Conjugate along with improved stability and immunogenicity.
- Fig 1 SEC-HP-RI profile of sized 19A PnPs (178 KDa) before DMAP treatment
- Fig.2 SEC-HP-RI profile of sized 19A PnPs (70 KDa) after 24 h (A) and 14.5 KDa after 72 h (B) DMAP treatment-degradation profiles
- Fig.3 SEC-HP-RI profiles of sized 19A PnPs (178 KDa; A) and time -dependent aggregation of CDAP-mediated activated polysaccharides (5B, 5C & 5D),5E (conjugate without 10 KDa DF) ,5F (conjugate with 10 KDa DF),5G-sized 19A/5H- Activated 19A(modified Ps:CDAP ratio 1:1 for 19A)conjugation method without using 10 KDa diafiltration step.
- CDAP-mediated activated polysaccharides 5B, 5C & 5D
- 5E conjuggate without 10 KDa DF
- 5F conjuggate with 10 KDa DF
- Fig.4 SEC-HP-RI profiles of sized 19F PnPs (157 KDa; A) and time -dependent aggregation of CDAP-mediated activated polysaccharides (B), 6C/6D(modified Ps:CDAP ratio 1:1 for 19F)conjugation method without using 10 KDa diafiltration step
- the inventors of present invention have observed that combined adsorption of polysaccharide-protein conjugates and use of polysaccharide to protein ratio greater than 1 : 1 results in i) percent adsorption of less than 55% for S.
- the polysaccharide was cultivated using a method as described in Patent WO2013088448A1, wherein said method comprises (a) providing an inoculum of a strain of bacteria expressing the CP; (b) cultivating the strain by fermentation at pH 7.2, wherein the rate of feed medium addition is equivalent to the rate of alkali mixture addition for maintaining a preset pH; c) fermenting the culture medium at 35-38°C under stirring at 50-150 RPM with an air flow rate of 0.1-0.5vvm.
- the polysaccharide was purified by the process described in Patent WO2012127485.
- Pn-Ps prepared by the instant process shows recovery of about 60 to 70%, wherein C-polysaccharide contamination reduction is of 1 to 5 fold as compared to the C-Ps content of post -Hydrophobic interaction chromatography (HIC) or pre ion exchange chromatography (IEC), protein contamination is less than 1% and nucleic acid contamination is less than 1%.
- HIC post -Hydrophobic interaction chromatography
- IEC pre ion exchange chromatography
- This process can purify polysaccharides with 80-90% less time consumption & 90% less cost when compared with CTAB/Alcohol based methods.
- improved percent adsorption between 75 to 95% can be obtained for S. pneumoniae conjugates by i) employing polysaccharide to protein ratio of about 0.8 to 1.4 ii) utilizing individual or separate adsorption for poorly adsorbing S. pneumoniae conjugates, iii) keeping lower pH during formulation.
- preferred polysaccharide to protein ratio is 1:1.
- said composition comprises of atleast 2 polysaccharide protein conjugates having polysaccharide selected from serotypes 1, 2, 3,4,5, 6A, 6B,7F, 8,9V,9F,9N, 12F, 14, 15B, 17F,18C, 19A ,19F,20,22F,23F,33F and 45
- individual mode of adsorption can be utilized for any S. pneumoniae serotype selected from of 2, 3, 4, 6A, 8, 9V, 9F, 9N, 12F, 15B, 17F, 18C, 20, 22F, 23F, 33F and 45, preferably for S. pneumoniae serotypes 6A, 9V, and 23F.
- the multivalent pneumococcal conjugate vaccine is 10 valent wherein S. pneumoniae serotypes 6 A, 9V, and 23F are individually adsorbed as a separate blend and then added to another blend comprising of a mixture of S. pneumoniae serotypes 1, 5, 6B, 7F, 14, 19A and 19F that have been adsorbed in a combined mode.
- the multivalent pneumococcal conjugate vaccine is 11, 13 , 15 , 16 or more valent wherein atleast one S. pneumoniae serotype selected from a group of 2, 3, 4, 6A, 8, 9V, 9F, 9N, 12F, 15B, 17F, 18C, 20, 22F, 23F, 33F and 45 is individually adsorbed or adsorbed in a smaller group as a separate blend and then added to another blend comprising of a mixture of S. pneumoniae serotypes 1, 5, 6B, 7F, 14, 19A and 19F that have been adsorbed in a combined mode.
- the multivalent pneumococcal conjugate vaccine is 16 valent wherein atleast one S. pneumoniae serotype selected from a group of 2, 3, 4, 6 A, 9V, 12F, 15B, 18C, and 23F is individually adsorbed or in smaller groups as a separate blend and then added to another blend comprising of a mixture of S. pneumoniae serotypes 1, 5, 6B, 7F, 14, 19A and 19F that have been adsorbed in a combined mode.
- a second embodiment of the instant invention is that aggregation in a multivalent pneumococcal polysaccharide-protein conjugate formulation can be completely prevented by i) utilizing a pH shift from neutral pH to acidic pH and ii) use of histidine-succinic acid buffer combination.
- said pH shift can occur from 6.8 to a pH selected from but not limited to 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8 and 5.9. More preferably from 6.8 to a pH selected from 5.4, 5.5, 5.6, 5.7 and 5.8.
- said Histidine-Succinic acid buffer system can have a concentration between lmM and 200 M.
- the concentration is preferably at least 1 mM (e.g. at most 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM etc.). More preferably the concentration of Histidine-Succinic acid buffer in the composition is between 10 mM and 40 mM.
- a third embodiment of the instant invention is that floccules or aggregate formation in pneumococcal bulk conjugates can be prevented by utilizing a rushton turbine flat blade impellers instead of a magnetically stirred, axial and radial type impellers in formulation vessels.
- the stability of an immunogenic composition of the invention is readily determined using standard techniques, which are well known and routine to those of skill in the art.
- an immunogenic composition is assayed for percent adsorption of conjugates, stability, aggregation, immunogenicity, particulate formation, protein (concentration) loss, and the like, by methods including, but not limited to, ELISA, light scattering, optical density, sedimentation velocity centrifugation, sedimentation equilibrium centrifugation, circular dichroism(CD), Lowry assay, bicinchoninic acid (BCA) assay, and the like.
- instant invention provides a novel ELISA that can directly quantify conjugated/bound polysaccharide without affecting antigenicity of conjugates in multivalent pneumococcal conjugate vaccines.
- the same can be utilized for quantification of unadsorbed conjugate content in formulation matrix as an indicating parameter for percent adsorption wherein the conjugates show more than 70% adsorption.
- said ELISA can employ a pre- assay step involving desorption of conjugate from alum adjuvant without impacting the antigenicity of the carrier protein as well as the conjugated Polysaccharide.More specifically, the dissolution of alum adsorbed conjugate samples is achieved using sodium hydroxide and citric acid.
- the carrier protein can be selected from a group of but not limited to CRM197, P4, diphtheria toxoid, tetanus toxoid, fragment C of tetanus toxoid, pertussis toxoid, protein D of H. influenzae, E. coli LT, E.
- coli ST and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA) , pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11 , protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD), particularly CRM197 or P4.
- OMPC outer membrane complex c
- PspA pneumococcal surface protein A
- PsaA pneumococcal surface adhesin A
- said multivalent composition can comprise of i) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having TT as carrier protein; or ii) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having DT as carrier protein; or iii) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having pneumococcal surface adhesin A (PsaA) as carrier protein; or iv) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having TT as carrier protein, atleast one polysaccharide protein having DT as carrier protein ; or v) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having TT as carrier protein,
- the preferred carrier protein conjugated to Serotype 3 is CRM-197
- Serotype 4 is TT or DT
- Serotype 18C is CRM197.
- Another embodiment of the present invention includes the use of PsaA as a carrier protein in the final formulation.
- the PsaA can also be used in the final formulation as an adjuvant.
- multivalent formulation of instant invention can comprise of a surfactant preferably polysorbate 20.
- the final concentration of the polysorbate 20 in formulation is 0.01% to 10% polysorbate 20 weight/volume of the formulation.
- the final concentration of the polysorbate 20 in the formulation is 0.01% polysorbate 20 weight/volume of the formulation.
- the final concentration of the polysorbate 20 in the formulation is 0.05% polysorbate 20 weight/volume of the formulation.
- the final concentration of the polysorbate 20 in the formulation is 0.1% polysorbate 20 weight/volume of the formulation.
- the final concentration of the polysorbate 20 in the formulation is 1.0% polysorbate 20 weight/volume of the formulation.
- the final concentration of the polysorbate 20 in the formulation is 10.0% polysorbate 20 weight/volume of the formulation.
- the present multivalent vaccine formulations can comprise of preservatives selected from a group of but not limited to mercurial preservatives (e.g. thimerosal), 2-phenoxy-ethanol, methyl parabens, propyl parabens and benzyl alcohol (or mixtures thereof).
- said multivalent pneumococcal polysaccharide-protein conjugate vaccine formulation preferably 10 or 16 valent can comprise of aluminium phosphate adsorbed conjugates, Histidine, Succinic acid, Sodium chloride, Polysorbate 20 and thiomersal.
- the vaccine composition of instant invention can comprise of a step of adding aluminium salt adjuvant at an amount of 20-375 ⁇ g, 20-300 ⁇ g, 20-200 ⁇ g, 25-150 ⁇ g of A1+++ per 0.5 ml dose.
- the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
- the preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect.
- Direct delivery of the compositions will generally be parenteral (e.g. injection, subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue).
- the compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications needles, and hyposprays.
- Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses).
- the vaccines of the present invention may be stored in solution or lyophilized, wherein the lyophilized vaccine composition of the instant invention can be given as 1, 5 or 10 dose formulation with a diluent containing aluminium phosphate gel and NaCl.
- Another embodiment of the present invention includes the use of Rushton Turbine Flat Blade Impeller in the formulation vessel (Refer Figure 5).
- Example 1 Fermentation Method comprises (a) providing inoculum of a strain of bacteria expressing the CP; (b) cultivating the strain by fermentation at pH 7.2, wherein the rate of feed medium addition is equivalent to the rate of alkali mixture addition for maintaining a preset pH; c) fermenting the culture medium at 35-38°C under stirring at 50-150 RPM with an air flow rate of 0-0.5 vvm.
- Example 2 Capsular polysaccharide purification
- Nuclease was added to the polysaccharide solution to achieve a final concentration of 8 U/ml of solution.
- the enzyme treatment was carried out at 370C, for 10 + 2 hrs with stirring.
- Ammonium sulphate was added to the nuclease treated polysaccharide solution to 50% saturation and incubated at 2 - 8°C for 12 + 2 hrs (except serotypes 5 and 4). The mixture was subjected to centrifugation. The pellet (precipitate) was discarded. The solution (-500 ml) is subjected to 100 kD diafiltration using NaCl followed by chilled WFI. This diafiltered solution containing polysaccharide with a buffer and high salt concentration was loaded on HIC column.
- the hydrophobic interaction chromatography column (300 ml) was equilibrated with 50% saturated ammonium sulphate buffer and the polysaccharide solution (500 ml) was then loaded onto the column in pH range 6 to 8, preferably at pH 6 to 7 pH. The column was further washed with the buffer containing 50% saturated ammonium sulphate. Under these conditions, the polysaccharide was recovered in the flow -through and equilibration wash from the column. The polysaccharide solution was then concentrated using a 100 KDa MWCO filter and then diafiltered with NaCl and Water for Injection (WFI).
- WFI Water for Injection
- the ion exchange chromatography column (300 ml)(strong anion exchanger) was equilibrated with 20 mM sodium phosphate buffer and the polysaccharide solution (500 ml) was then loaded onto the column in pH range 6 to 8, preferably at pH 6.5 to 7.5 pH. The column was further washed with buffer. The adsorbed polysaccharides were eluted with step gradient elution using 1.0 M NaCl (various polysaccharides were eluted at different ionic strengths of NaCl).
- the polysaccharide solution was then concentrated using a 100 KDa MWCO filter and then diafiltered with Water for Injection (WFI).
- WFI Water for Injection
- the diafiltered polysaccharide solution was filtered through a 0.22 ⁇ membrane filter into polypropylene bottles.
- the purified polysaccharide was stored frozen at -20+50C.
- a homogenizer (Microfluidics) apparatus was used to reduce the molecular weight of the polysaccharide before the activation step. For 19A size reduction was done at 24-28 KPSI, whereas for 19F size reduction was done at 26-30 KPSI wherein the number of passes was about 1 to 3.
- the sized polysaccharide was diafiltered and concentrated followed by 0.22 ⁇ filtration. The sized polysaccharide was then subjected to HPSEC-RI for estimation of average molecular weight.
- Example 4 General Conjugation process Conjugation of polysaccharide to carrier protein was carried out using CDAP conjugation method of Lees et al (Vaccine 26: 190-198, 1996). Mechanically size reduced polysaccharides (except for 6A which was used in native form or sized depending on size of 6A) were dissolved in NaCl 2M. CDAP (in acetonitrile) from a lOOmg/ml stock solution was added to the polysaccharide solution as per polysaccharide:CDAP ratio . Approximate 1 minute later, 2M NaOH was added to obtain the specific activation pH. The activation of the polysaccharide was performed at this pH during 4-10 minutes at 22°C.
- CRM- 197 (the quantity depend on initial Ps/Protein ratio) was added to the activated polysaccharide and the coupling reaction was performed at the specific pH for 3-8 hr depending on serotype. The reaction was then quenched with glycine for 1 hr at 220C, and overnight at 120C. The conjugates were then purified by 300kDa to 500kDa diafilteration followed by lOOkDa diafilteration. Further the polysaccharide and protein content of the purified 0.22um filtered conjugates were determined.
- Antigen content and percent adsorption was determined using modified Sandwich ELISA.
- the conventional Sandwich ELISA has been modified with respect to following test conditions/assay conditions and thereby has following advantageous attributes- i. to quantify conjugated Polysaccharide in the presence of 9 other conjugated antigens in a 10 valent vaccine ii. to elute all ten conjugate from aluminum phosphate gel where adsorption is more than 80 % iii. wherein capture of conjugate occurs even if it is at pH 9 without damaging the conjugate antigenicity
- Antigen content and percent adsorption was determined using ELISA as per the protocol given below:
- Capture Antibody anti -carrier protein antibody
- PCV16 Pneumococcal Conjugate Vaccine - 16 Valent
- the active ingredients of the vaccine are conjugated to atleast one carrier protein selected from CRM197, TT and DT.
- the active ingredients of the vaccine are conjugated to atleast one carrier protein selected from CRM197,TT and DT.
- Sized PnPs in reaction solution was treated with DMAP in a ratio of 1:1.5 and checked for its degradation profile by SEC-HP-RI.
- activated PnPs was subjected to 10 KDa diafiltration using 2M NaCl to remove the DMAP formed from reaction solution before conjugation with CRM 197 .
- activated PnPs was subjected to 10 KDa diafiltration using 2M NaCl to remove the DMAP formed from reaction solution before conjugation with CRM 197 .
- Refer Figures 3E (conjugate without 10 KDa DF) & 3F (conjugate with 10 KDa DF) which shows the SEC-HP-RI profile of conjugate using 10 KDa diafiltered activated PnPs and CRM 197 .
- diafilteration step was adversely affecting conjugate yield resulting in a 30% -40% decrease in overall yield.
- modified Ps:CDAP ratios(l:l for 19A and 1:0.8 for 19F) were found to prevent degradation and aggregation for both 19A and 19F PnPs.
- An additional advantage of using modified ratios was that it was devoid of a 10 KDa diafiltration step thus ensuring minimum loss in overall yield.
- Ratio PnPs/CDAP 1.0:1.5 1.0:1.0 1.0:1.5 1.0:0.8
- Ps/CRM 197 1.0:1.5 1.0:1.0 1.0:1.5 1.0:1.0 pH a :pH c :pH q 9.0:9.0:9.0 9.0:9.0:9.0 9.5:9.5:9.5 9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5:9.5
- modified Polysaccharide:CDAP ratio, CDAP activation time and initial polysaccharide:protein ratio were found to minimize 4-dimethylamino-pyridine mediated degradation of sized polysaccharide during activation and also prevented subsequent polysaccharide- polysaccharide aggregation thereby improving final conjugate characteristics with respect to free polysaccharide content.
Abstract
The present invention provides methods for preparation of stable multivalent pneumococcal polysaccharide-protein conjugate vaccine formulations. Instant stable formulations show optimal percent adsorption for each conjugate wherein, aggregation can be prevented by employing i) Individual or separate adsorption for conjugates that otherwise show lower percent adsorption by combined adsorption ii) Histidine-Succinic acid buffer system along with shift in pH from neutral pH to acidic pH iii) a polysaccharide to protein ratio between 0.5 to about 1.4 iv) a six-bladed Rushton type turbine impeller in formulation vessels.
Description
Title:
Methods for improving the adsorption of polysaccharide -protein conjugates and multivalent vaccine formulation obtained thereof.
Background of the invention Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis in children. Recent estimates of child deaths caused by 5 pneumoniae range from 0.7 - 1.0 million every year worldwide. In 2000, about 14· 5 million episodes of serious pneumococcal disease (uncertainty range 11· 1-18-0 million) were estimated to occur. Pneumococcal disease caused about 0.826 million deaths (0.58 -0.926 M) in children aged 1-59 months, of which 0.091 million (0.063-0.1 M) were in HIV-positive and 0.735 million (0.51-0.82 M) in HIV-negative children.
The multivalent pneumococcal polysaccharide vaccines that have been licensed for many years have proved valuable in preventing pneumococcal disease in adults, particularly, the elderly and those at high-risk. However, infants and young children respond poorly to unconjugated pneumococcal polysaccharides. The pneumococcal conjugate vaccine, Prevnar®, containing the 7 most frequently isolated serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) causing invasive pneumococcal disease in young children and infants at the time, was first licensed in the United States in February 2000.
Further Prevnar™ 13 (Wyeth) is an approved vaccine that contains conjugates of polysaccharides from serotypes 6A, 6B, 19A, 19F in addition to 1, 3, 4, 5, 7F, 9V, 14, 18C and 23F. Synflorix™ (GSK) is another approved vaccine that provides protection against 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F as well as cross protection against 19A & 6 A.
Vaccine formulations must generally be stable and be of uniform consistency to accommodate the need for a long shelf-life and the use of multiple dose containers. Vaccines based on proteins, including polysaccharide -protein conjugates, are subject to protein aggregation and precipitation which can result in an effective lower total concentration of the vaccine due to the unavailability of the precipitated protein product. Polysaccharide -protein conjugate vaccines, in particular, appear to have a stronger tendency to aggregate than the carrier protein alone (See Berti et al, 2004, Biophys J 86:3-9). The choice of formulation for a polysaccharide -protein conjugate vaccine can greatly affect protein aggregation. See Ho et al., 2001, Vaccine 19:716-725.
Inspite of several existing multivalent pneumococcal polysaccharide -protein conjugate vaccine compositions being developed worldwide, there is an ongoing need in the art for vaccine formulations that provide high adsorption of individual conjugates and free from the aggregation/precipitation of immunogenic compositions having polysaccharide -protein conjugates.
The adjuvants traditionally used in such multivalent pneumococcal vaccines have been aluminium salts such as aluminium hydroxide and aluminium phosphate. Many other experimental adjuvants are known, however adsorption to aluminium salts remains the most common vaccine adjuvant formulation. Although their use is widespread, aluminium salts may not always be compatible with particular antigens thereby resulting in significant variations with respect to percent adsorption of polysaccharide-protein conjugate on alum or the antigenicity.
Immunological properties and stability of conjugate vaccine candidate adsorbed on aluminium adjuvants depends on various parameters like i) antigenicity of each antigen, ii) type of carrier protein used for conjugation, and iii) the type of adjuvant used. Most importantly, the extent of adsorption of antigen on the adjuvant has been previously reported to be one of the key parameters to demonstrate the lot to lot consistency of the formulation process and its possible impact on the efficacy of the vaccine product. Further, percent adsorption of polysaccharide-protein conjugates may drop further on storage of the formulation or under the adverse situations like temperature excursions. Refer 54th meeting of the WHO Expert Committee on Biological Standardization, Recommendations for the production & control of Pneumococcal conjugate vaccines, 17-21 November 2003; Carl E. Frasch, Session IV: Conjugate Vaccines; Vaccine Technology II;Portugal. 2008.
As per European regulatory agency (EMEA) guidelines for pneumococcal polysaccharide-protein conjugates, completeness of adsorption (% unbound conjugate) should be considered as a crucial quality control parameter along with alum content, sterility, identity and free polysaccharide content. Refer Assessment Report for Synflorix 2009, Procedure No. EMEA/H/C/000973. WHO recommends maximizing adsorption for alum-precipitated antigens(e.g. diphtheria and tetanus toxoid) wherein at least 80% of the antigens in these vaccines be adsorbed.
During manufacturing of polysaccharide-protein conjugate, formulate can comprise of aggregates of polysaccharide-polysaccharide type, protein-protein type or polysaccharide-protein type. Such aggregations are also observed in the finished product leading to rejection of 4% to 10% of filled vials of polysaccharide-protein conjugate(s) vaccine, thereby affecting the stability and efficacy of the conjugate vaccine.
Given the above discussed limitations with respect to the aggregation and stability of the polysaccharide and its conjugate, there remains a distinct need for reducing aggregation and stabilizing said polysaccharide across downstream processing to the final formulation stage of Pneumococcal Conjugate Vaccine Manufacturing.
Summary of the Invention:
The invention provides improvements in the stability of vaccines which include aluminium salts, and in particular methods for minimizing aggregation and improvements in percent adsorption of individual conjugates in a multivalent pneumococcal polysaccharide -protein conjugate vaccines. The inventors of present invention have observed that combined adsorption of polysaccharide -protein conjugates and use of polysaccharide to protein ratio greater than 1 : 1 results in i) percent adsorption of less than 55% for S. pneumoniae conjugates for serotypes 6A, 9V and 23F and ii) percent adsorption from about 80% to 90% for remaining serotype conjugates, thereby failing to achieve complete adsorption for an individual serotype conjugate for a given multivalent pneumococcal conjugate formulation. Also it was observed that vaccine formulation prepared by using pH between 6.8 to 7.0 resulted in an aggregation of about 4 to 10% and lower adsorption.
The instant invention relates to a method for the preparation of a stable multivalent pneumococcal polysaccharide-protein conjugate vaccine formulation, showing optimal adsorption between 75 to 99% for each conjugate wherein, in said method, aggregation is prevented by employing at least one of: a. Individual or separate adsorption for conjugates that otherwise show relative lower adsorption by combined adsorption; b. Histidine-Succinic acid buffer system along with shift in pH from neutral pH to acidic pH; c. A polysaccharide to protein ratio between 0.6 to about 1.4; and/or d. The use of a six bladed Rushton type turbine impeller in the formulation vessel. The instant invention also discloses a method for preparing polysaccharide-protein conjugates with improved immunogenicity and less free polysaccharide content for Streptococcus pneumoniae polysaccharides containing phosphodiester linkage, particularly 19A, 19F, 6A and 6B. Said conjugation process minimizes cyanylation agent by-product mediated degradation of sized polysaccharide and prevents subsequent polysaccharide -polysaccharide aggregation thereby stabilizing labile polysaccharides. A key to reduced aggregation can be attributed to the use of a sized polysaccharide in the range of 100 - 200 KDa, and polysaccharide to CDAP (Cyanylation agent) ratio in the range of (1):(0.8 - 1).
The immunogenic composition prepared as per the instant invention provides reduced aggregation between Polysaccharide - Polysaccharide, and Polysaccharide - Protein Conjugate along with improved stability and immunogenicity.
Figures:
Fig 1: SEC-HP-RI profile of sized 19A PnPs (178 KDa) before DMAP treatment
Fig.2: SEC-HP-RI profile of sized 19A PnPs (70 KDa) after 24 h (A) and 14.5 KDa after 72 h (B) DMAP treatment-degradation profiles Fig.3: SEC-HP-RI profiles of sized 19A PnPs (178 KDa; A) and time -dependent aggregation of CDAP-mediated activated polysaccharides (5B, 5C & 5D),5E (conjugate without 10 KDa DF) ,5F (conjugate with 10 KDa DF),5G-sized 19A/5H- Activated 19A(modified Ps:CDAP ratio 1:1 for 19A)conjugation method without using 10 KDa diafiltration step.
Fig.4: SEC-HP-RI profiles of sized 19F PnPs (157 KDa; A) and time -dependent aggregation of CDAP-mediated activated polysaccharides (B), 6C/6D(modified Ps:CDAP ratio 1:1 for 19F)conjugation method without using 10 KDa diafiltration step
Fig.5: Six bladed Rushton type turbine Flat Blade Impeller
Fig 6: Adsorption protocol
Detailed Description: It is an object of the invention to provide improvements in the stability of vaccines which include aluminium salts and, in particular, methods for minimizing aggregation and improvements in percent adsorption of individual conjugates in a multivalent pneumococcal polysaccharide -protein conjugate vaccines. The inventors of present invention have observed that combined adsorption of polysaccharide-protein conjugates and use of polysaccharide to protein ratio greater than 1 : 1 results in i) percent adsorption of less than 55% for S. pneumoniae conjugates for Serotypes 6 A, 9V and 23F and ii) percent adsorption from about 80% to 90% for remaining serotype conjugates, thereby failing to achieve complete adsorption for an individual serotype conjugate for a given multivalent pneumococcal conjugate formulation. Also it was observed that vaccine formulation prepared by using pH between 6.8 to 7.0 resulted in an aggregation of about 4 to 10% and lower adsorption. The polysaccharide was cultivated using a method as described in Patent WO2013088448A1, wherein said method comprises (a) providing an inoculum of a strain of bacteria expressing the CP; (b) cultivating the strain by fermentation at pH 7.2, wherein the rate of feed medium addition is equivalent to the rate of alkali mixture addition for maintaining a preset pH; c) fermenting the culture medium at 35-38°C under stirring at 50-150 RPM with an air flow rate of 0.1-0.5vvm. The polysaccharide was purified by the process described in Patent WO2012127485. Pn-Ps prepared by the instant process shows recovery of about 60 to 70%, wherein C-polysaccharide contamination reduction is of 1 to 5 fold as compared to the C-Ps content of post -Hydrophobic interaction
chromatography (HIC) or pre ion exchange chromatography (IEC), protein contamination is less than 1% and nucleic acid contamination is less than 1%. The said process has been carried out at Research, Pilot and commercial scale.
This process can purify polysaccharides with 80-90% less time consumption & 90% less cost when compared with CTAB/Alcohol based methods.
According to one important embodiment of the instant invention, improved percent adsorption between 75 to 95% can be obtained for S. pneumoniae conjugates by i) employing polysaccharide to protein ratio of about 0.8 to 1.4 ii) utilizing individual or separate adsorption for poorly adsorbing S. pneumoniae conjugates, iii) keeping lower pH during formulation. According to one aspect of first embodiment, preferred polysaccharide to protein ratio is 1:1.
According to a second aspect of first embodiment, said composition comprises of atleast 2 polysaccharide protein conjugates having polysaccharide selected from serotypes 1, 2, 3,4,5, 6A, 6B,7F, 8,9V,9F,9N, 12F, 14, 15B, 17F,18C, 19A ,19F,20,22F,23F,33F and 45
According to a third aspect of first embodiment, individual mode of adsorption can be utilized for any S. pneumoniae serotype selected from of 2, 3, 4, 6A, 8, 9V, 9F, 9N, 12F, 15B, 17F, 18C, 20, 22F, 23F, 33F and 45, preferably for S. pneumoniae serotypes 6A, 9V, and 23F.
According to a preferred aspect of first embodiment, the multivalent pneumococcal conjugate vaccine is 10 valent wherein S. pneumoniae serotypes 6 A, 9V, and 23F are individually adsorbed as a separate blend and then added to another blend comprising of a mixture of S. pneumoniae serotypes 1, 5, 6B, 7F, 14, 19A and 19F that have been adsorbed in a combined mode.
According to another preferred aspect of first embodiment, the multivalent pneumococcal conjugate vaccine is 11, 13 , 15 , 16 or more valent wherein atleast one S. pneumoniae serotype selected from a group of 2, 3, 4, 6A, 8, 9V, 9F, 9N, 12F, 15B, 17F, 18C, 20, 22F, 23F, 33F and 45 is individually adsorbed or adsorbed in a smaller group as a separate blend and then added to another blend comprising of a mixture of S. pneumoniae serotypes 1, 5, 6B, 7F, 14, 19A and 19F that have been adsorbed in a combined mode.
According to yet another preferred aspect of first embodiment, the multivalent pneumococcal conjugate vaccine is 16 valent wherein atleast one S. pneumoniae serotype selected from a group of 2, 3, 4, 6 A, 9V, 12F, 15B, 18C, and 23F is individually adsorbed or in smaller groups as a separate blend and then added to another blend comprising of a mixture of S. pneumoniae serotypes 1, 5, 6B, 7F, 14, 19A and 19F that have been adsorbed in a combined mode.
A second embodiment of the instant invention is that aggregation in a multivalent pneumococcal polysaccharide-protein conjugate formulation can be completely prevented by i) utilizing a pH shift from neutral pH to acidic pH and ii) use of histidine-succinic acid buffer combination.
According to one aspect of second embodiment, said pH shift can occur from 6.8 to a pH selected from but not limited to 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8 and 5.9. More preferably from 6.8 to a pH selected from 5.4, 5.5, 5.6, 5.7 and 5.8.
According to another aspect of second embodiment, said Histidine-Succinic acid buffer system can have a concentration between lmM and 200 M. The concentration is preferably at least 1 mM (e.g. at most 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM etc.). More preferably the concentration of Histidine-Succinic acid buffer in the composition is between 10 mM and 40 mM.
A third embodiment of the instant invention is that floccules or aggregate formation in pneumococcal bulk conjugates can be prevented by utilizing a rushton turbine flat blade impellers instead of a magnetically stirred, axial and radial type impellers in formulation vessels. The stability of an immunogenic composition of the invention is readily determined using standard techniques, which are well known and routine to those of skill in the art. For example, an immunogenic composition is assayed for percent adsorption of conjugates, stability, aggregation, immunogenicity, particulate formation, protein (concentration) loss, and the like, by methods including, but not limited to, ELISA, light scattering, optical density, sedimentation velocity centrifugation, sedimentation equilibrium centrifugation, circular dichroism(CD), Lowry assay, bicinchoninic acid (BCA) assay, and the like.
In a preferred embodiment, instant invention provides a novel ELISA that can directly quantify conjugated/bound polysaccharide without affecting antigenicity of conjugates in multivalent pneumococcal conjugate vaccines. The same can be utilized for quantification of unadsorbed conjugate content in formulation matrix as an indicating parameter for percent adsorption wherein the conjugates show more than 70% adsorption. Preferably, said ELISA can employ a pre- assay step involving desorption of conjugate from alum adjuvant without impacting the antigenicity of the carrier protein as well as the conjugated Polysaccharide.More specifically, the dissolution of alum adsorbed conjugate samples is achieved using sodium hydroxide and citric acid. The carrier protein can be selected from a group of but not limited to CRM197, P4, diphtheria toxoid, tetanus toxoid, fragment C of tetanus toxoid, pertussis toxoid, protein D of H. influenzae, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA) , pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3
and BVH-11 , protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD), particularly CRM197 or P4.
According to a preferred embodiment, said multivalent composition can comprise of i) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having TT as carrier protein; or ii) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having DT as carrier protein; or iii) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having pneumococcal surface adhesin A (PsaA) as carrier protein; or iv) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having TT as carrier protein, atleast one polysaccharide protein having DT as carrier protein ; or v) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having TT as carrier protein, atleast one polysaccharide protein having pneumococcal surface adhesin A(PsaA) as carrier protein ; or vi) atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having DT as carrier protein, atleast one polysaccharide protein having pneumococcal surface adhesin A (PsaA) as carrier protein.
In another embodiment, the preferred carrier protein conjugated to Serotype 3 is CRM-197, Serotype 4 is TT or DT and Serotype 18C is CRM197.
Another embodiment of the present invention includes the use of PsaA as a carrier protein in the final formulation. The PsaA can also be used in the final formulation as an adjuvant.
In certain embodiments, multivalent formulation of instant invention can comprise of a surfactant preferably polysorbate 20. In certain embodiments, the final concentration of the polysorbate 20 in formulation is 0.01% to 10% polysorbate 20 weight/volume of the formulation. In yet other embodiments, the final concentration of the polysorbate 20 in the formulation is 0.01% polysorbate 20 weight/volume of the formulation. In other embodiments, the final concentration of the polysorbate 20 in the formulation is 0.05% polysorbate 20 weight/volume of the formulation. In yet other embodiments, the final concentration of the polysorbate 20 in the formulation is 0.1% polysorbate 20
weight/volume of the formulation. In another embodiment, the final concentration of the polysorbate 20 in the formulation is 1.0% polysorbate 20 weight/volume of the formulation. In yet another embodiment, the final concentration of the polysorbate 20 in the formulation is 10.0% polysorbate 20 weight/volume of the formulation. The present multivalent vaccine formulations can comprise of preservatives selected from a group of but not limited to mercurial preservatives (e.g. thimerosal), 2-phenoxy-ethanol, methyl parabens, propyl parabens and benzyl alcohol (or mixtures thereof).
According to a preferred embodiment of present invention, said multivalent pneumococcal polysaccharide-protein conjugate vaccine formulation, preferably 10 or 16 valent can comprise of aluminium phosphate adsorbed conjugates, Histidine, Succinic acid, Sodium chloride, Polysorbate 20 and thiomersal.
The vaccine composition of instant invention can comprise of a step of adding aluminium salt adjuvant at an amount of 20-375 μg, 20-300μg, 20-200μg, 25-150μg of A1+++ per 0.5 ml dose.
Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect. Direct delivery of the compositions will generally be parenteral (e.g. injection, subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue). The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications needles, and hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses).
Preferably, the vaccines of the present invention may be stored in solution or lyophilized, wherein the lyophilized vaccine composition of the instant invention can be given as 1, 5 or 10 dose formulation with a diluent containing aluminium phosphate gel and NaCl.
Another embodiment of the present invention includes the use of Rushton Turbine Flat Blade Impeller in the formulation vessel (Refer Figure 5).
Examples:
Example 1: Fermentation Method comprises (a) providing inoculum of a strain of bacteria expressing the CP; (b) cultivating the strain by fermentation at pH 7.2, wherein the rate of feed medium addition is equivalent to the rate of alkali mixture addition for maintaining a preset pH; c) fermenting the culture medium at 35-38°C under stirring at 50-150 RPM with an air flow rate of 0-0.5 vvm.
Example 2: Capsular polysaccharide purification
S. Pneumoniae Capsular Polysaccharide Serotype 19F Purification (HIC followed by IEC)
5L clarified broth from the fermenter cultures of S. pneumoniae serotype 19F was concentrated and diafiltered to 500 ml using a 100 KDa MWCO membrane. Diafiltration was accomplished using 25 mM sodium phosphate buffer at neutral pH followed by diafiltration with water for injection (WFI).
Nuclease was added to the polysaccharide solution to achieve a final concentration of 8 U/ml of solution. The enzyme treatment was carried out at 370C, for 10 + 2 hrs with stirring.
Ammonium sulphate was added to the nuclease treated polysaccharide solution to 50% saturation and incubated at 2 - 8°C for 12 + 2 hrs (except serotypes 5 and 4). The mixture was subjected to centrifugation. The pellet (precipitate) was discarded. The solution (-500 ml) is subjected to 100 kD diafiltration using NaCl followed by chilled WFI. This diafiltered solution containing polysaccharide with a buffer and high salt concentration was loaded on HIC column.
The hydrophobic interaction chromatography column (300 ml) was equilibrated with 50% saturated ammonium sulphate buffer and the polysaccharide solution (500 ml) was then loaded onto the column in pH range 6 to 8, preferably at pH 6 to 7 pH. The column was further washed with the buffer containing 50% saturated ammonium sulphate. Under these conditions, the polysaccharide was recovered in the flow -through and equilibration wash from the column. The polysaccharide solution was then concentrated using a 100 KDa MWCO filter and then diafiltered with NaCl and Water for Injection (WFI).
The ion exchange chromatography column (300 ml)(strong anion exchanger) was equilibrated with 20 mM sodium phosphate buffer and the polysaccharide solution (500 ml) was then loaded onto the column in pH range 6 to 8, preferably at pH 6.5 to 7.5 pH. The column was further washed with buffer. The adsorbed polysaccharides were eluted with step gradient elution using 1.0 M NaCl (various polysaccharides were eluted at different ionic strengths of NaCl).
The polysaccharide solution was then concentrated using a 100 KDa MWCO filter and then diafiltered with Water for Injection (WFI).
The diafiltered polysaccharide solution was filtered through a 0.22 μ membrane filter into polypropylene bottles. The purified polysaccharide was stored frozen at -20+50C.
The above process was also utilized for serotypes 4, 6A, 6B, 7F, 9V, 10A, 14, 18C, 19A, 19F, & 23F.
Results:
C-Polysaccharide post HIC & post Ion exchange chromatography was estimated by H1/P31 NMR spectra. The process resulted in 2-3 fold reduction in contaminants content.
Example 3: Sizing of Polysaccharides
A homogenizer (Microfluidics) apparatus was used to reduce the molecular weight of the polysaccharide before the activation step. For 19A size reduction was done at 24-28 KPSI, whereas for 19F size reduction was done at 26-30 KPSI wherein the number of passes was about 1 to 3. The sized polysaccharide was diafiltered and concentrated followed by 0.22 μ filtration. The sized polysaccharide was then subjected to HPSEC-RI for estimation of average molecular weight.
Example 4: General Conjugation process Conjugation of polysaccharide to carrier protein was carried out using CDAP conjugation method of Lees et al (Vaccine 26: 190-198, 1996). Mechanically size reduced polysaccharides (except for 6A which was used in native form or sized depending on size of 6A) were dissolved in NaCl 2M. CDAP (in acetonitrile) from a lOOmg/ml stock solution was added to the polysaccharide solution as per polysaccharide:CDAP ratio . Approximate 1 minute later, 2M NaOH was added to obtain the specific activation pH. The activation of the polysaccharide was performed at this pH during 4-10 minutes at 22°C. CRM- 197 (the quantity depend on initial Ps/Protein ratio) was added to the activated polysaccharide and the coupling reaction was performed at the specific pH for 3-8 hr depending on serotype. The reaction was then quenched with glycine for 1 hr at 220C, and overnight at 120C. The conjugates were then purified by 300kDa to 500kDa diafilteration followed by lOOkDa diafilteration. Further the polysaccharide and protein content of the purified 0.22um filtered conjugates were determined.
Table 1- Serotype specific conjugation reaction parameter variations for 10 serotypes:
9.5:9. 9.5:9.5:9. 9.5:9.5: 9.5:9. 9.5:9. 9.5:9. 9.5:9.5: 09:0 9.5:9. 9.5:9. pHa:pHc:pHq
5:9.5 5 9.5 5:9.5 5:9.5 5:9.5 9.5 9:09 5:9.5 5:9.5
Free Ps (%) < 1 6.3 2.63 1.57 < 1 ND 2.53 1.5 1.07 2.59
Free protein (%) ND 1.54 2.2 ND ND ND ND ND 2.2 ND
Mol. Size
70.56 74.93 67.67 76.52 75.68 72.75 76.59 78.1 74.35 72.91 Distribution (%)
Table 2: Comparison in percent adsorption of individual serotypes of PCV-10 formulations
and two- blend approach of formulation process.
whereas for the two blend approach the percent adsorption was >70% for all serotypes. Table 3: Effect of pH shift on aggregation behavior of PCV-10 formulations
No aggregates were found for the pH 5.6-5.8 for PCVIO formulation when compared with that of pH
The polysaccharide to protein ratio (1:1) was found to have advantageous effect on the extent of adsorption of Pneumococcal conjugate in formulation.
Table 4: Effect of Ps to CRM197 ratio on adsorption of pneumococcal conjugate (serotype 6A) in Formulation
The effect of polysaccharide to protein ratio was observed on adsorption of conjugates. 6A conjugates which had Ps to protein ratio of >1.5 showed poor adsorption. Most of the serotypes used in the PCV-10 formulation contained Ps: Pr ratio in the range of 0.6-1.3 which resulted in optimum adsorption of various serotypes including serotype 6 A. Based on the adsorption data achieved for 6 A serotype and PCV-10 formulation experience, while formulating 16 valent it can be extrapolated that Ps: Pr ratio of remaining 6 conjugates in the similar range could ensure consistent adsorption of all 16 serotype conjugates.
Table 5: Specifications of Rushton Turbine Flat Blade Impeller for different vessels
Example 6: ELISA Protocol
Antigen content and percent adsorption was determined using modified Sandwich ELISA.
The conventional Sandwich ELISA has been modified with respect to following test conditions/assay conditions and thereby has following advantageous attributes- i. to quantify conjugated Polysaccharide in the presence of 9 other conjugated antigens in a 10 valent vaccine
ii. to elute all ten conjugate from aluminum phosphate gel where adsorption is more than 80 % iii. wherein capture of conjugate occurs even if it is at pH 9 without damaging the conjugate antigenicity
Antigen content and percent adsorption was determined using ELISA as per the protocol given below:
Day 1
1. The plates were coated with Capture Antibody (anti -carrier protein antibody) followed by incubation for 0.5- 2 hrs at 35 ± 5 °C
2. plates were washed 3-6 times using ELISA plate washer
3. plates were blocked with blocking buffer (3% BSA in IX PBS and tris buffer for serotypes 14 and 9V) followed by incubation for 1.5 - 2.5 hrs at 30 ±5°C
4. plates were washed 3 times using ELISA plate washer
5. samples were added into the plates
6. plates were incubated overnight at 5+3 °C
Day 2
1. plates were allowed to attain room temperature
2. primary antibody was added after washing of the plates 3 times followed by incubation at RT for 25+2 for 30 mins followed by washing.
3. secondary antibody was added and followed by incubation at 25+2 for 30 minutes.
4. substrate TMB was added followed by incubation at RT for 15-20 mins in dark
5. stop solution was added
6. plates were read at 450nm.
Procedure for dissolution of sample without harming the epitope of carrier protein
An appropriate quantity of 0.5 M- 2M NaoH was added to 2 ml of vaccine sample. Said sample was subjected to vortex gently untill the solution became clear. The pH of the solution was adjusted from 9-12 till the solution became clear. pH of the solution was brought back to 6-7.4 using 0.5M to 2M citric acid. Said solution was subjected to centrifugation (dissolved samples) at 3000 to 6000 x g for 5 min and supernatant was collected for the testing.
Table 6
Serotype 6A 3.39 83 4.6 68.3
Serotype 6B 8.01 80 6.8 85
Serotype 7F 4.72 97 4.18 84.4
Serotype 9V 4.61 80 3.95 70.4
Serotype 14 5.11 94 5.06 92.5
Serotype 19A 4.42 94 3.65 91.8
Serotype 19F 4.35 82 4.14 81.4
Serotype 23F 3.66 72 4.03 75
Example 7: Pneumococcal Conjugate Vaccine - 10 Valent (PCV10)
Table 7- PCV10 composition:
Active ingredients are conjugated with Carrier protein CRM197
Example 8: Formulation 1 (PCV16)
Pneumococcal Conjugate Vaccine - 16 Valent (PCV16) Table 8- PCV16 composition "I"
* The active ingredients of the vaccine are conjugated to atleast one carrier protein selected from CRM197, TT and DT.
** Added only in multi-dose presentation
Example 9: Formulation 1 (PCV16)
Table 9- PCV16 composition "Π"
* The active ingredients of the vaccine are conjugated to atleast one carrier protein selected from CRM197,TT and DT.
** Added only in multi-dose presentation
Example 10:
I) Degradation of sized PnPs (19A, 19F, 6A and 6B)in presence of DMAP:
Sized PnPs in reaction solution was treated with DMAP in a ratio of 1:1.5 and checked for its degradation profile by SEC-HP-RI.
Results:
It was observed that only 19A PnPs undergoes degradation in presence of DMAP whereas other phosphodiester containing PnPs (19F, 6A & 6B) remain intact. Refer Figures 1 (without DMAP) & 2 which show the DMAP-mediated degradation of PnPs.
To minimize such degradation of 19A, activated PnPs was subjected to 10 KDa diafiltration using 2M NaCl to remove the DMAP formed from reaction solution before conjugation with CRM197.
II) Degradation and aggregation of sized PnPs (19A,19F,6A and 6B)in presence of CDAP:
Sized PnPs in reaction solution was treated with CDAP in the ratio of 1:1.5 during activation. It was observed that 50% DMAP is generated as by-product(measured by RP-HPLC)which leads to degradation of PnPs as well as aggregation between activated PnPs after certain time of activation (refer Table 2)Degradation and aggregation was checked by SEC-HP-RI profile. Refer Figures 3A (without CDAP), 3B, 3C & 3D (for 19A) & 4A (without CDAP) 4B (for 19F) which show the CDAP- mediated aggregation & degradation of PnPs.
Table 10- Factors responsible for polysaccharide-polysaccharide cross linking & aggregation
Effect of "duration of CDAP activation" on formation of "polysaccharide -polysaccharide aggregates"
Mw of sized/modified Activated modified
Serotypes PnPs (KDa) [SEC- PnPs (KDa) [SEC-HP- Effects
HP-RI] RI]
No aggregation between
6A 432 435
activated polysaccharides
No aggregation between
6B 131 131
activated polysaccharides
Aggregation between
254 (after 20 min)
activated polysaccharides
Aggregation between
19A 178 487 (after 60 min)
activated polysaccharides
Aggregation between
1173 (after 120 min)
activated polysaccharides
Aggregation between
19F 157 205 (after 30 min)
activated polysaccharides
III) Prevention of degradation and aggregation of sized PnPs (19A and 19F) having PnPs:CDAP of 1:1.5 by employing diafiltration step:
To minimize such degradation and aggregation of 19A, activated PnPs was subjected to 10 KDa diafiltration using 2M NaCl to remove the DMAP formed from reaction solution before conjugation with CRM197.Refer Figures 3E (conjugate without 10 KDa DF) & 3F (conjugate with 10 KDa DF) which shows the SEC-HP-RI profile of conjugate using 10 KDa diafiltered activated PnPs and CRM197. However diafilteration step was adversely affecting conjugate yield resulting in a 30% -40% decrease in overall yield.
IV) Prevention of degradation and aggregation of sized PnPs (19A and 19F) by reducing ratio of PnPs:CDAP to 1:1 (19A) and 1:0.8 (19F) without employing diafiltration step
Sized PnPs in reaction solution was treated with CDAP in the ratios of 1:1 and 1:0.8 for 19A and 19F respectively and checked for its degradation and aggregation profile by SEC-HP-RI. Results:
It was observed that modified Ps:CDAP ratios(l:l for 19A and 1:0.8 for 19F)were found to prevent degradation and aggregation for both 19A and 19F PnPs. Refer Figures 3 (G & H for 19A) & 4 (C &
D for 19F). An additional advantage of using modified ratios was that it was devoid of a 10 KDa diafiltration step thus ensuring minimum loss in overall yield.
It was observed that in case of serotype 19 A, when "duration of CDAP activation" was more than 10 min, it was resulting in cross linking of activated polysaccharide to activated polysaccharide ultimately leading to formation of "polysaccharide -polysaccharide aggregates".
Further in case of serotype 19F, when "duration of CDAP activation" was more than 20 min, it was resulting in cross linking of activated polysaccharide to activated polysaccharide ultimately leading to formation of "polysaccharide -polysaccharide aggregates" .Further the duration of conjugation reaction was found to be more than the duration required for cross linking of activated polysaccharide to activated polysaccharide thereby resulting in formation of aggregates. Refer Figures 3 & 4.
However for other serotypes like 6APnPs and 6BPnPs, such cross linking was not observed.
Example 11:
Preparation of conjugates: PnPsl9A & PnPsl9F
Conjugation of polysaccharide to carrier protein was carried out using CDAP conjugation method of Lees et al (Vaccine 26: 190-198, 1996) with following modifications: i) For preparing 19A conjugate, using a polysaccharide to CDAP ratio of 1:1 at 22°C with a period of activation of 4 min and using a polysaccharide to protein ratio of 1 : 1 ii) For preparing 19F conjugate, using a polysaccharide to CDAP ratio of 1:0.8 at 22°C with a period of activation of 9 to 10 min and a polysaccharide to protein ratio of 1:1 for 19F. Table 10- Comparison of conjugate results for 19A and 19F with "traditional CDAP conjugation method" (Lot 1) and "improved conjugation method" (Lot 2)
CRM Cone (mg/ml) 20 20 20 20
Initial Ratio
Ps/CRM197 1.0:1.5 1.0:1.0 1.0:1.5 1.0:1.0 pHa:pHc:pHq 9.0:9.0:9.0 9.0:9.0:9.0 9.5:9.5:9.5 9.5:9.5:9.5
II) Final Conjugate Characteristics
Conjugates 19A conjugate 19F conjugate
Batch Number Lot l Lot 2 Lot l Lot 2
Final Ratio
PnPs/CRM197 0.53 0.86 0.52 0.96
CRM197/PnPs 1.88 1.16 1.92 1.04
Free PnPs (%) 1.9 1.5 1.7 1.07
Free CRM197 (%) ND ND ND 2.2
Avg. Mol. Size SEC-
1081 853 994 847
HP-I)(KDa)
Avg.Mol. size
8212 6012 6393 4773
(UV/RI MALS) (KDa)
Results:
It was observed that modified Polysaccharide:CDAP ratio, CDAP activation time and initial polysaccharide:protein ratio, were found to minimize 4-dimethylamino-pyridine mediated degradation of sized polysaccharide during activation and also prevented subsequent polysaccharide- polysaccharide aggregation thereby improving final conjugate characteristics with respect to free polysaccharide content.
The improved conjugation method employed for preparing 19 A & 19F conjugates resulted in conjugates that did not show any phosphomonoester signal in respective conjugate profiles(31P Proton
NMR)which indicated that modified conjugation method was found to be effective in preventing hydrolysis of polysaccharides across conjugation reactions.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
Claims
1. A method for the preparation of a stable multivalent pneumococcal polysaccharide -protein conjugate vaccine formulation, showing optimal adsorption for each conjugate wherein, in said method, aggregation is prevented by employing at least one of: i. Individual or separate adsorption for conjugates that otherwise show relative lower adsorption by combined adsorption; ii. histidine-Succinic acid buffer system along with shift in pH from neutral pH to acidic pH; iii. a polysaccharide to protein ratio between 0.5 to about 1.4; and/or iv. the use of a rushton type turbine impeller in the formulation vessel.
2. The method as claimed in Claim 1, wherein the adsorption is in the range of 75 - 99%.
3. A multivalent pneumococcal conjugate vaccine composition prepared according to claim 1; wherein said composition comprises of atleast 2 polysaccharide protein conjugates having polysaccharide selected from serotypes 1, 2, 3,4,5, 6 A, 6B,7F, 8,9V,9F,9N, 12F, 14, 15B, 17F,18C, 19A ,19F,20,22F,23F,33F and 45.
4. A multivalent pneumococcal conjugate vaccine composition prepared according to claim 3; wherein said composition comprises of atleast 10 polysaccharide protein conjugates having polysaccharide from serotypes 1, 5, 6A, 6B,7F, 9V, 14, 19A ,19F and 23F.
5. A multivalent pneumococcal conjugate vaccine composition prepared according to claim 3; wherein said composition comprises of atleast 16 polysaccharide protein conjugates having polysaccharide from serotypes 1,2,3,4,5,6A,6B,7F,9V,12F,14,15B,18C,19A,19F and 23F.
6. The composition as claimed in Claim 1, wherein carrier protein is one or more selected from the group consisting of CRM 197, Pneumococcal surface adhesin A (PsaA), diphtheria toxoid(DT), tetanus toxoid(TT), fragment C of tetanus toxoid, pertussis toxoid, protein D of H. influenzae, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA) , pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11 , protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine
serum albumin (BSA) and purified protein derivative of tuberculin (PPD), particularly CRM197,tetanus toxoid, diphtheria toxoid or PsaA.
7. The composition as claimed in claim 1, wherein said multivalent composition comprises of i. atleast one polysaccharide protein conjugate having CRM197 as carrier protein , atleast one polysaccharide protein having TT as carrier protein; or ii. atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having DT as carrier protein; or iii. atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having pneumococcal surface adhesin A(PsaA) as carrier protein; or iv. atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having TT as carrier protein, atleast one polysaccharide protein having DT as carrier protein ; or v. atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having TT as carrier protein, atleast one polysaccharide protein having pneumococcal surface adhesin A (PsaA) as carrier protein ; or vi. atleast one polysaccharide protein conjugate having CRM 197 as carrier protein , atleast one polysaccharide protein having DT as carrier protein, atleast one polysaccharide protein having pneumococcal surface adhesin A (PsaA) as carrier protein.
8. The composition as claimed in Claim 3, wherein serotype 3 is preferably conjugated to CRM- 197, serotype 18C is preferably conjugated to CRM197 and Serotype 4 can be conjugated to TT or DT.
9. The method as claimed in claim 1, wherein said histidine-succinic acid buffer has a concentration between lmM and 200mM, preferably between lOmM and 40mM, more preferably 20mM.
10. The method as claimed in Claim 1, wherein the shift in pH for the histidine-succinic buffer occurs preferably from 6.8 to a pH between 5.6 and 5.8.
11. The method as claimed in claim 1, wherein said method comprises of following steps:
i. adding polysaccharide molecules to a protein carrier in a predetermined ratio, preferably 0.8 to 1.4 and more preferably 1:1, and at a predetermined pH, preferably between 5.2 and 5.9, to obtain a plurality of distinct polysaccharide -protein conjugates; ii. adsorbing individually on a first aluminium salt adjuvant in a histidine-succinic acid buffer having a predetermined concentration, a first predetermined set of polysaccharide -protein conjugates selected from said plurality of polysaccharide - protein conjugates obtained in step (i), wherein the amount of said aluminium salt adjuvant ranges from 20-375 μg of A1+++ per 0.5 ml dose of the final vaccine formulation; wherein the set of polysaccharide -protein conjugates comprises of 6A, 9V and 23F. iii. adsorbing in a combined mode on a second aluminium salt adjuvant in a histidine- succinic acid buffer having a predetermined concentration, a second predetermined set of polysaccharide -protein conjugates selected from said plurality of polysaccharide -protein conjugates obtained in step (i), wherein the amount of said aluminium salt adjuvant ranges from 20-375 μg of A1+++ per 0.5 ml dose of the final vaccine formulation; wherein the set of polysaccharide -protein conjugates comprises of 1, 5, 6B, 7F, 14, 19A and 19F. iv. blending said first adsorbed set obtained in step (ii) with said second adsorbed set obtained in step (iii), and optionally adding surfactant preferably Polysorbate-20 and/or atleast one preservative selected from the group consisting of mercurial preservatives such as Thimerosal, 2-phenoxy-ethanol, methyl parabens, propyl parabens, benzyl alcohol and mixtures thereof, with the help of a Rushton turbine flat blade impeller to obtain a stable multivalent pneumococcal polysaccharide -protein conjugate vaccine formulation.
A method for reducing polysaccharide -polysaccharide aggregation across sizing and conjugation reactions, particularly for Streptococcus pneumoniae polysaccharide containing a phosphodiester linkage between repeat units covalently linked to a carrier protein using cyanylation chemistry, thereby providing improved immunogenicity and stability for each conjugate , wherein said method comprises of: i) reacting polysaccharide having average size between 130 KDa and 190 KDa with a cyanylation agent in a ratio between 1:0.8 and 1:1 at about 22°C to about 25°C for a
period of about 4 min to about 10 min, resulting in a cyanate -activated polysaccharide; and
ii) contacting cyanate-activated polysaccharide with protein in a ratio of 1 : 1 at a pH of about 9 to about 9.5 for a period of 3hr to 5 hr followed by quenching with glycine; wherein said conjugation process minimizes cyanylation agent by-product mediated degradation of sized polysaccharide and prevents subsequent polysaccharide - polysaccharide aggregation resulting in a conjugate having minimum free polysaccharide content.
13. The method of claim 12, wherein the polysaccharide comprising a phosphodiester linkage between repeat units is derived from one of Streptococcus pneumoniae serotype 19 A, 19F, 6 A or 6B.
14. The method as claimed in Claim 13, wherein the polysaccharide is Streptococcus pneumoniae serotype 19A polysaccharide and cyanylation agent is l-cyano-4-dimethyl- amino-pyridinium-tetrafluoroborate mixed in a ratio of 1 : 1 at 22°C for a period of 4 minutes resulting in a cyanate-activated polysaccharide.
15. The method as claimed in Claim 13, wherein the polysaccharide is Streptococcus pneumoniae serotype 19F polysaccharide and Cynalating agent isl-cyano-4-dimethyl- amino-pyridinium-tetrafluoroborate mixed in a ratio of 1:0.8 at 22°C for a period of 8-10 minutes resulting in a cyanate-activated polysaccharide.
16. The method as claimed in Claim 14 or 15, wherein the said conjugate has free polysaccharide content of less than 1.6%.
17. The method of Claim 2, wherein percent adsorption is measured by a Sandwich ELISA particularly comprising of a step of dissolution of alum adsorbed conjugate samples using sodium hydroxide and citric acid.
18. The composition as claimed in any of the preceding claims, wherein PsaA is used as an adjuvant.
19. The composition as claimed in any of the preceding claims, which further includes the step of storing said conjugate vaccine formulation obtained in a lyophilized state, until administration as a 1, 5 or 10 dose formulation with a diluent containing aluminium phosphate gel and NaCl.
Priority Applications (18)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020187000352A KR20180030498A (en) | 2015-06-08 | 2016-06-03 | A method for improving adsorption of a polysaccharide-protein conjugate and a polyvalent vaccine preparation obtained therefrom |
| KR1020237045298A KR20240007690A (en) | 2015-06-08 | 2016-06-03 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
| AU2016276269A AU2016276269B2 (en) | 2015-06-08 | 2016-06-03 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
| EA201792470A EA201792470A1 (en) | 2015-06-08 | 2016-06-03 | METHODS TO IMPROVE THE ADSORPTION OF POLYSACCHARIDE-PROTEIN CONJUGATES AND A COMPOUND OF POLYVALENT VACCINE RECEIVED FROM THEM |
| CA2988366A CA2988366C (en) | 2015-06-08 | 2016-06-03 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof. |
| NZ738090A NZ738090B2 (en) | 2016-06-03 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof | |
| CN201680040596.5A CN107847571A (en) | 2015-06-08 | 2016-06-03 | Method for the absorption of the polyvaccine preparaton that improves proteinpolysaccharide conjugate and its acquisition |
| MYPI2017704714A MY187461A (en) | 2015-06-08 | 2016-06-03 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
| EP16806975.5A EP3302542A4 (en) | 2015-06-08 | 2016-06-03 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
| US15/580,653 US10729780B2 (en) | 2015-06-08 | 2016-06-03 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
| MX2017015985A MX2017015985A (en) | 2015-06-08 | 2016-06-03 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof. |
| JP2017564108A JP7258463B2 (en) | 2015-06-08 | 2016-06-03 | Methods for Improving Adsorption of Polysaccharide-Protein Conjugates and Multivalent Vaccine Formulations Resulting Therefrom |
| BR112017026343-2A BR112017026343A2 (en) | 2015-06-08 | 2016-06-03 | methods for improving the adsorption of protein-polysaccharide conjugates and thus obtained multivalent vaccine formulation |
| IL256118A IL256118B (en) | 2015-06-08 | 2017-12-05 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
| ZA2017/08325A ZA201708325B (en) | 2015-06-08 | 2017-12-07 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
| CONC2017/0012628A CO2017012628A2 (en) | 2015-06-08 | 2017-12-07 | Methods to improve adsorption of polysaccharide-protein conjugates and a multivalent vaccine formulation obtained therewith |
| PH12017502244A PH12017502244A1 (en) | 2015-06-08 | 2017-12-07 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
| SA517390495A SA517390495B1 (en) | 2015-06-08 | 2017-12-10 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN2185MU2015 | 2015-06-08 | ||
| IN2185/MUM/2015 | 2015-06-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2016199003A1 true WO2016199003A1 (en) | 2016-12-15 |
Family
ID=57503115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IB2016/053265 WO2016199003A1 (en) | 2015-06-08 | 2016-06-03 | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
Country Status (18)
| Country | Link |
|---|---|
| US (1) | US10729780B2 (en) |
| EP (1) | EP3302542A4 (en) |
| JP (2) | JP7258463B2 (en) |
| KR (2) | KR20240007690A (en) |
| CN (1) | CN107847571A (en) |
| AU (1) | AU2016276269B2 (en) |
| BR (1) | BR112017026343A2 (en) |
| CA (1) | CA2988366C (en) |
| CO (1) | CO2017012628A2 (en) |
| EA (1) | EA201792470A1 (en) |
| IL (1) | IL256118B (en) |
| MX (1) | MX2017015985A (en) |
| MY (1) | MY187461A (en) |
| PE (1) | PE20180460A1 (en) |
| PH (1) | PH12017502244A1 (en) |
| SA (1) | SA517390495B1 (en) |
| WO (1) | WO2016199003A1 (en) |
| ZA (1) | ZA201708325B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106822884A (en) * | 2016-12-28 | 2017-06-13 | 北京民海生物科技有限公司 | A kind of preparation method of polyvalent pneumococcal capsular polysaccharide conjugate vaccine |
| CN108524926A (en) * | 2018-06-29 | 2018-09-14 | 康希诺生物股份公司 | A kind of formulation compositions of multivalent pneumococcal combined vaccine and its application |
| JP2020509002A (en) * | 2017-02-24 | 2020-03-26 | メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． | Pneumococcal conjugate vaccine preparation |
| US10946086B2 (en) | 2015-05-04 | 2021-03-16 | Pfizer Inc. | Group B Streptococcus polysaccharide-protein conjugates, methods for producing conjugates, immunogenic compositions comprising conjugates, and uses thereof |
| US11116828B2 (en) | 2017-12-06 | 2021-09-14 | Merck Sharp & Dohme Corp. | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| US11642406B2 (en) | 2018-12-19 | 2023-05-09 | Merck Sharp & Dohme Llc | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10729780B2 (en) * | 2015-06-08 | 2020-08-04 | Serum Institute Of India Private Ltd. | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
| US11951165B2 (en) | 2016-12-30 | 2024-04-09 | Vaxcyte, Inc. | Conjugated vaccine carrier proteins |
| US10729763B2 (en) | 2017-06-10 | 2020-08-04 | Inventprise, Llc | Mixtures of polysaccharide-protein pegylated compounds |
| CN111065388A (en) * | 2017-09-07 | 2020-04-24 | 默沙东公司 | Pneumococcal polysaccharides and their use in immunogenic polysaccharide-carrier protein conjugates |
| AU2020286360A1 (en) * | 2019-06-05 | 2021-12-23 | Merck Sharp & Dohme Llc | An immunogenic serotype 35B pneumococcal polysaccharide-protein conjugate and conjugation process for making the same |
| US20210070890A1 (en) | 2019-09-06 | 2021-03-11 | Serum Institute Of India Private Limited | Method for obtaining purified bacterial polysaccharides |
| CN113607939A (en) * | 2021-08-05 | 2021-11-05 | 艾美探索者生命科学研发有限公司 | Method for measuring adsorption rate of various combined stock solutions of pneumococcal polysaccharide combined vaccine |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5623057A (en) * | 1991-01-28 | 1997-04-22 | Merck & Co., Inc. | Pneumococcal polysaccharide conjugate vaccine |
| US20070110762A1 (en) * | 2003-08-06 | 2007-05-17 | Department Of Health And Human Services, Govt. of the United States of America | Process for preparing polysaccharide-protein conjugate vaccines |
| US20100209450A1 (en) * | 2007-06-26 | 2010-08-19 | Ralph Leon Biemans | Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates |
| US20110195086A1 (en) * | 2010-02-09 | 2011-08-11 | Caulfield Michael J | 15-valent pneumococcal polysaccharide-protein conjugate vaccine composition |
| US20140377302A1 (en) * | 2012-01-30 | 2014-12-25 | Serum Institute Of India Ltd. | Immunogenic composition |
| EP2865392A1 (en) * | 2012-06-20 | 2015-04-29 | SK Chemicals Co., Ltd. | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2289546A3 (en) * | 2003-01-30 | 2011-03-30 | Novartis Vaccines and Diagnostics S.r.l. | Injectable vaccines against multiple meningococcal serogroups |
| US8119146B2 (en) * | 2005-04-18 | 2012-02-21 | Angelica Medina-Selby | Expressing hepatitis B virus surface antigen for vaccine preparation |
| CN101378778B (en) * | 2005-12-22 | 2013-02-06 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine comprising streptococcus pneumoniae capsular polyaccharide conjugates |
| BRPI0620418A2 (en) * | 2005-12-23 | 2011-11-08 | Glaxosmithkline Biolog Sa | method for immunizing a human patient against a disease, uses at least two and at least seven, ten, eleven, thirteen or fourteen conjugates and vaccines, and |
| TW200806315A (en) * | 2006-04-26 | 2008-02-01 | Wyeth Corp | Novel formulations which stabilize and inhibit precipitation of immunogenic compositions |
| US20130273098A1 (en) * | 2010-12-10 | 2013-10-17 | Jeffrey T. Blue | Novel formulations which mitigate agitation-induced aggregation of immunogenic compositions |
| DK2885007T3 (en) * | 2012-08-16 | 2018-12-03 | Pfizer | Methods for glycoconjugation and compositions |
| CN103893751B (en) * | 2014-03-26 | 2016-04-20 | 天津康希诺生物技术有限公司 | A kind of pneumococal polysaccharide Protein Conjugation vaccine and preparation method thereof |
| US10729780B2 (en) * | 2015-06-08 | 2020-08-04 | Serum Institute Of India Private Ltd. | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof |
-
2016
- 2016-06-03 US US15/580,653 patent/US10729780B2/en active Active
- 2016-06-03 CN CN201680040596.5A patent/CN107847571A/en active Pending
- 2016-06-03 EA EA201792470A patent/EA201792470A1/en unknown
- 2016-06-03 EP EP16806975.5A patent/EP3302542A4/en active Pending
- 2016-06-03 PE PE2017002539A patent/PE20180460A1/en unknown
- 2016-06-03 WO PCT/IB2016/053265 patent/WO2016199003A1/en active Application Filing
- 2016-06-03 AU AU2016276269A patent/AU2016276269B2/en active Active
- 2016-06-03 JP JP2017564108A patent/JP7258463B2/en active Active
- 2016-06-03 KR KR1020237045298A patent/KR20240007690A/en active Application Filing
- 2016-06-03 MY MYPI2017704714A patent/MY187461A/en unknown
- 2016-06-03 CA CA2988366A patent/CA2988366C/en active Active
- 2016-06-03 MX MX2017015985A patent/MX2017015985A/en unknown
- 2016-06-03 BR BR112017026343-2A patent/BR112017026343A2/en active Search and Examination
- 2016-06-03 KR KR1020187000352A patent/KR20180030498A/en not_active Application Discontinuation
-
2017
- 2017-12-05 IL IL256118A patent/IL256118B/en unknown
- 2017-12-07 PH PH12017502244A patent/PH12017502244A1/en unknown
- 2017-12-07 CO CONC2017/0012628A patent/CO2017012628A2/en unknown
- 2017-12-07 ZA ZA2017/08325A patent/ZA201708325B/en unknown
- 2017-12-10 SA SA517390495A patent/SA517390495B1/en unknown
-
2021
- 2021-08-06 JP JP2021129726A patent/JP2021185152A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5623057A (en) * | 1991-01-28 | 1997-04-22 | Merck & Co., Inc. | Pneumococcal polysaccharide conjugate vaccine |
| US20070110762A1 (en) * | 2003-08-06 | 2007-05-17 | Department Of Health And Human Services, Govt. of the United States of America | Process for preparing polysaccharide-protein conjugate vaccines |
| US20100209450A1 (en) * | 2007-06-26 | 2010-08-19 | Ralph Leon Biemans | Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates |
| US20110195086A1 (en) * | 2010-02-09 | 2011-08-11 | Caulfield Michael J | 15-valent pneumococcal polysaccharide-protein conjugate vaccine composition |
| US20140377302A1 (en) * | 2012-01-30 | 2014-12-25 | Serum Institute Of India Ltd. | Immunogenic composition |
| EP2865392A1 (en) * | 2012-06-20 | 2015-04-29 | SK Chemicals Co., Ltd. | Polyvalent pneumococcal polysaccharide-protein conjugate composition |
Non-Patent Citations (1)
| Title |
|---|
| See also references of EP3302542A4 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10946086B2 (en) | 2015-05-04 | 2021-03-16 | Pfizer Inc. | Group B Streptococcus polysaccharide-protein conjugates, methods for producing conjugates, immunogenic compositions comprising conjugates, and uses thereof |
| CN106822884A (en) * | 2016-12-28 | 2017-06-13 | 北京民海生物科技有限公司 | A kind of preparation method of polyvalent pneumococcal capsular polysaccharide conjugate vaccine |
| JP2020509002A (en) * | 2017-02-24 | 2020-03-26 | メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． | Pneumococcal conjugate vaccine preparation |
| JP7254707B2 (en) | 2017-02-24 | 2023-04-10 | メルク・シャープ・アンド・ドーム・エルエルシー | Pneumococcal conjugate vaccine formulation |
| US11116828B2 (en) | 2017-12-06 | 2021-09-14 | Merck Sharp & Dohme Corp. | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| US11850278B2 (en) | 2017-12-06 | 2023-12-26 | Merck Sharp & Dohme Llc | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
| CN108524926A (en) * | 2018-06-29 | 2018-09-14 | 康希诺生物股份公司 | A kind of formulation compositions of multivalent pneumococcal combined vaccine and its application |
| CN108524926B (en) * | 2018-06-29 | 2021-06-29 | 康希诺生物股份公司 | Preparation combination of multivalent pneumococcal conjugate vaccine and application thereof |
| US11642406B2 (en) | 2018-12-19 | 2023-05-09 | Merck Sharp & Dohme Llc | Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CO2017012628A2 (en) | 2018-03-28 |
| BR112017026343A2 (en) | 2019-11-19 |
| AU2016276269B2 (en) | 2021-01-28 |
| KR20180030498A (en) | 2018-03-23 |
| CN107847571A (en) | 2018-03-27 |
| MY187461A (en) | 2021-09-23 |
| IL256118B (en) | 2021-12-01 |
| US20180161445A1 (en) | 2018-06-14 |
| EP3302542A1 (en) | 2018-04-11 |
| KR20240007690A (en) | 2024-01-16 |
| JP2021185152A (en) | 2021-12-09 |
| NZ738090A (en) | 2023-09-29 |
| EA201792470A1 (en) | 2018-10-31 |
| ZA201708325B (en) | 2019-05-29 |
| CA2988366A1 (en) | 2016-12-15 |
| JP2018516962A (en) | 2018-06-28 |
| PH12017502244A1 (en) | 2018-06-11 |
| SA517390495B1 (en) | 2023-03-23 |
| IL256118A (en) | 2018-02-28 |
| EP3302542A4 (en) | 2019-04-10 |
| JP7258463B2 (en) | 2023-04-17 |
| CA2988366C (en) | 2021-12-07 |
| MX2017015985A (en) | 2018-08-15 |
| PE20180460A1 (en) | 2018-03-06 |
| US10729780B2 (en) | 2020-08-04 |
| AU2016276269A1 (en) | 2018-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2016276269B2 (en) | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof | |
| US10245311B2 (en) | Adjuvanting meningococcal factor H binding protein | |
| CN104302315B (en) | Immunogenic composition and preparation method thereof | |
| AU2019201131B2 (en) | Meningococcus vaccines | |
| WO2015144031A1 (en) | Pneumococcus polysaccharide protein conjugated vaccine and preparation method therefor | |
| US10596246B2 (en) | Adjuvanted combinations of meningococcal factor H binding proteins | |
| CN103083652B (en) | A kind of meningococcal polysaccharides combined vaccine with Heterobifunctional reagents as cross structure and preparation method thereof | |
| CN102438649A (en) | Combinations of meningococcal factor h binding protein and pneumococcal saccharide conjugates | |
| JP6764021B2 (en) | Neisseria Meningitidis Vaccine | |
| JP2017522320A (en) | Modified meningococcal FHBP polypeptide | |
| US20220211859A1 (en) | Conjugate production | |
| OA19863A (en) | Methods for improving the adsorption of polysaccharide-Protein conjugates and multivalent vaccine formulation obtained thereof. | |
| TW201818960A (en) | A process for preparing a Hib conjugate vaccine using PRP with a lowered molecular weigh | |
| TW201932140A (en) | Methods for improving the adsorption of polysaccharide-protein conjugates and multivalent vaccine formulation obtained thereof | |
| EA040838B1 (en) | STABLE POLYVALENT VACCINE COMPOSITION AND METHOD FOR ITS PRODUCTION |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16806975 Country of ref document: EP Kind code of ref document: A1 |
|
| ENP | Entry into the national phase |
Ref document number: 2988366 Country of ref document: CA |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 11201710058W Country of ref document: SG |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 002539-2017 Country of ref document: PE |
|
| WWE | Wipo information: entry into national phase |
Ref document number: NC2017/0012628 Country of ref document: CO Ref document number: 12017502244 Country of ref document: PH Ref document number: 201792470 Country of ref document: EA |
|
| ENP | Entry into the national phase |
Ref document number: 2017564108 Country of ref document: JP Kind code of ref document: A |
|
| WWE | Wipo information: entry into national phase |
Ref document number: MX/A/2017/015985 Country of ref document: MX |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| ENP | Entry into the national phase |
Ref document number: 20187000352 Country of ref document: KR Kind code of ref document: A |
|
| ENP | Entry into the national phase |
Ref document number: 2016276269 Country of ref document: AU Date of ref document: 20160603 Kind code of ref document: A |
|
| REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112017026343 Country of ref document: BR |
|
| REG | Reference to national code |
Ref country code: BR Ref legal event code: B01E Ref document number: 112017026343 Country of ref document: BR Free format text: COMO FORAM SOLICITADOS 2 SERVICOS ATRAVES DA PETICAO 870180006949 E, DE ACORDO COM A RESOLUCAO NO189/2017 DEVEM SER PAGAS RETRIBUICOES ESPECIFICAS PARA CADA UM DOS SERVICOS SOLICITADOS, SE FAZ NECESSARIA A COMPLEMENTACAO DO PAGAMENTO. |
|
| ENP | Entry into the national phase |
Ref document number: 112017026343 Country of ref document: BR Kind code of ref document: A2 Effective date: 20171206 |